The cytochrome P450 s comprise a large gene superfamily that encodes over 500 distinct heme-thiolate proteins that catalyze the oxidation of drugs and numerous other compounds in the body. It is of considerable interest in the pharmaceutical and other fields to identify cytochrome P450 s and the role they play in the metabolism of individual compounds. Cytochrome P450 s are heme—containing enzymes that strongly absorb at a wavelength of 450 nm when the heme is bound to a molecule of carbon monoxide. They are most well known for their ability to catalyze the metabolism of a wide variety of drugs, xenobiotics, carcinogens, mutagens and pesticides, and they are also involved in catalyzing reactions that make or degrade cholesterol, steroids, and other lipids. The reactions performed by these enzymes are generally oxidations, hydroxylations, acetylations, and demethylations. Mutations in cytochrome P450 s or abnormal expression levels can cause a number of human diseases such as glaucoma and breast cancer. Cytochrome P450 s are also involved in the metabolism of a number of vitamins, such as Vitamin A (retinoic acid) [White et. al. (1996) J. Biol. Chem. November, 22: 271(47): 29922-7; WO/97/49815; WO 01/44443] and Vitamin D [Jones, G. et. al. (1999) July; 140(7):3303-10; Dilworth F J, et. al. (1995) Jul. 14; 270(28); 16766-74. In particular, cytochrome P450 s, CYP27 and CYP24, are involved in Vitamin D3 and D2 metabolism. Vitamin D3 and D2, both seco-steroids, are metabolized into their active forms by CYP27 and are then further metabolized by CYP24. CYP24 is a mitochondrial cytochrome P450. that has previously been characterized. For example, isolated human CYP24 was published in Chen et al. (Isolation and expression of human 1,25-dihydroxyvitamin D3 24-hydroxylase cDNA. Proc Natl Acad Sci USA 1993 May 15; 90(10):4543-7). In Chen et al. it was reported that the human 24-hydroxylase 1539 base pair open reading frame encoded a 513 amino acid sequence, 90% homologous to rat CYP24. Mouse CYP24 was characterized in Yoshimura et al. (Molecular cloning of 25-hydroxyvitamin D-3 24-hydroxylase (Cyp-24) from mouse kidney: its inducibility by vitamin D-3. Biochim Biophys Acta 1995 Oct. 17; 1264(1):26-8).
The vitamin D metabolic pathway is part of a vital endocrine system that is highly regulated at certain stages and produces metabolites that control the secretion of the parathyroid gland hormones. 1α,25(OH)2D3, a hormone produced in the vitamin D pathway, regulates phosphate and calcium levels in the blood which in turn control bone mass, the state of bones, and affect cellular differentiation in the skin and the immune system. In the vitamin D pathway, cytochrome P450 s introduce functional groups by hydroxylation usually at positions 1, 25, and 24 of the steroid.
The metabolism of vitamin D begins with 25-hydroxlyation of vitamin D3 or D2 in the liver to 25(OH)D3. 25(OH)D3 and a second metabolite, 1α,25(OH)2D3, are converted to 24,25(OH)2D3 and 1,24,25(OH)3D3 by CYP24, a mitochondrial P450 involved in the vitamin D pathway. CYP24 is induced by 1,25(OH)2D3 and is found in the kidney as well as other vitamin D target tissues such as the parathyroid cells, keratinocytes, osteoblasts, and enteroctyes.
There are a number Vitamin D related medical conditions. More information on Vitamin D conditions can be found in the Proceedings of the Workshop on Vitamin D (Walter de Gruyter publishing, Berlin), proceedings 1 to 11. For instance, vitamin D deficiency has been related to the following:                (i) in the parathyroid—hyper- and hypo-parathyroidism, Osudohypo-parathyroidism, Secondary hyperparathyroidism;        (ii) in the pancreas—diabetes;        (iii) in the thyroid—medullary carcinoma;        (iv) in the skin—psoriasis;        (v) in the lung—sarcoidosis and tuberculosis;        (vi) in the kidney—chronic renal disease, glomerulonephritis, IgA nephropathy, membraneous nephropathy, glomerulosclerosis, nephrosis, renal insufficiency, hypophosphtatemic VDRR, vitamin D dependent rickets;        (vii) in the bone—anticonvulsant treatment, fibrogenisis imperfecta ossium, osteitits fibrosa cystica, osteomalacia, hypocicemia, osteporosis, osteopenia, osteosclerosis, renal osteodytrophy, rickets;        (viii) in the intestine—glucocorticoid antagonism, idopathic hypercalcemia, malabsorption syndrome, steatorrhea, tropical sprue;        (ix) in the prostate—cancer; and        (x) in the breast—cancer.        
More common conditions related to vitamin D or vitamin D metabolite deficiency are obesity problems, hyperhoshatemic turmoral calcinosis, sarcoidosis, tuberculosis, primary hyperparathyroidism, vitamin D dependent rickets type II, cholestatic or paremchymal liver disease.
Excess levels of Vitamin D can be toxic and can cause conditions such as hyperglycemia and mental deficiency. Such conditions usually present themselves upon excess ingestion of Vitamin D.
Since CYP24 is involved in maintaining Vitamin D homeostasis and is implicated in the development of these diseases, it is important to understand how CYP24 activity is and can be modulated in vivo and in vitro. For this, there is a need for a cellular model system that stably expresses CYP24. Such a model system would be especially useful in in vitro drug development studies. To date, there has been no stable cell line expressing CYP24. Cell lines that only generate transient expression are unsuitable for drug development assays. Some cell lines may also not be able to produce active protein (i.e. protein that is folded properly and able to perform a function, the function of native CYP24 such as catalysis). In the absence of a stable cell line, one cannot attribute reduced CYP24 activity to the effects of a candidate inhibitor when it is also possible that the cells merely have lost their ability to express CYP24. There is also a need for a cell model system that not only expresses CYP24 but expresses an active form of the peptide (e.g. that can fold properly and has endogenous CYP24 activities). There is also a need for a cell model system that allows recombinant CYP24 to be active within the cell.